This invention relates to a process for determining the carcinogenicity of a material or the presence of cancer-inducing factors in an environment.
A wide variety of materials, having very diverse chemical natures, are known to be capable of inducing cancer in man and other animals. In some cases, it has been found that materials which have been produced and used on a vast scale for decades have carcinogenic properties, so that millions of people have been exposed to such carcinogens before their carcinogenic nature was discovered. For example, benzene, which is commonly used in industry and in the laboratory as a solvent, has recently been found to be a carcinogen.
Increasing attention is now being paid to detecting carcinogenic properties of materials or environments before substantial numbers of persons are exposed thereto. Unfortunately, testing the carcinogenic character of materials poses a number of formidable problems. Known carcinogens include a large number of chemical compounds and mixtures having a wide variety of chemical structures, and the present state of knowledge as to how carcinogens cause cancer is such that not even experts in the field can estimate with any degree of confidence whether a specific compound will be a carcinogen, and if so, how potent a carcinogen. Consequently, every compound to which a substantial number of persons will be exposed must be tested for its carcinogenic character, and indeed, carcinogen testing tests have already been carried out on tens of thousands of compounds. Testing for carcinogenic character is further hampered by the long incubation periods, which can range from weeks to decades, between exposure to the carcinogen and the onset of the resultant cancer. Furthermore, carcinogens can be absorbed into the body in various ways, for example by mouth, through the lungs, or through the skin, and may cause cancer at a wide variety of different sites, often far removed from the locus at which the carcinogen enters the body. Some carcinogens will only cause cancer at a single site and there is no way of predicting, except in the case of compounds closely related to a known carcinogen at which site a suspected carcinogen under test will produce cancer. Thus, screening of a large number of possible carcinogens frequently involves elaborate dissection of large numbers of test animals, which greatly increases the costs of carcinogen testing.
Futhermore, since one does not know how long the incubation period will be before a carcinogen induces a cancer, it is necessary to minutely examine test animals for very small tumors after varying incubation times, and even skilled workers may miss very small tumors which might indicate carcinogenic character in the material under test.
There is thus a need for a carcinogen screening test which is cheap, can be rapidly applied to a large number of compounds, which will give an indication of the carcinogenic character of the test material regardless of the site at which that material exerts its carcinogenic character, and which does not depend upon elaborate visual inspection of a large number of tissues from test animals to visually detect tumors therein. Moreover, in view of the long incubation periods of many carcinogens, it is highly desirable that such a carcinogen screening test be able to give an indication of carcinogenic properties in a test material before macroscopically-visible tumors appear in the test animals, since this greatly reduces the duration and cost of such a carcinogen screening test. No prior art carcinogen screening test meets all these requirements. In particular, most prior art tests are limited in the variety of tumors which will give a positive indication in the test.
The present invention provides a carcinogen screening test which meets the above requirements.
In the aforementioned parent application Ser. No. 554,439, it is disclosed that a cancer-associated phosphoprotein can be isolated from the plasma of mammals suffering from any of a wide variety of cancers. This cancer-associated phosphoprotein ("cancer marker protein") has the following characteristics:
(a) not being precipitated by 30% saturated aqueous ammonium sulfate solution at 25.degree. C.;
(b) having a molecular weight of approximately 60,000;
(c) being precipitated from aqueous solution by 3.3% streptomycin sulfate;
(d) having substantially no autophosphorylation activity but being phosphorylated with adenosine triphosphate in the presence of an exogenous protein kinase;
(e) having substantially no protein kinase activity;
(f) having the capacity to liberate ribonucleic acid from cell nuclei; and
(g) being substantially free of albumin. In addition, unlike previously detected oncofetal proteins, the cancer-associated phosphoprotein is not present in the blood of healthy pregnant females.
The cancer-associated phosphoprotein is prepared by the following procedure:
(a) separating from the plasma of a mammal suffering from cancer the fraction of plasma protein which is not precipitated by 30% saturated aqueous ammonium sulfate solution;
(b) dispersing this fraction of plasma protein in a buffer and dialyzing the resultant protein solution against the buffer;
(c) separating the fraction of the dialyzed protein having a molecular weight of about 60,000; and
(d) removing substantially all albumin from the 60,000 molecular weight fraction.
It is further disclosed in the aforementioned application Ser. No. 554,439 (the entire disclosure of which is herein incorporated by reference) that the purified protein preparation prepared in the above manner can be introduced into the bloodstream of a mammal, thereby inducing in the mammal antibodies to the cancer-associated phosphoprotein, and these antibodies can be extracted in conventional ways to yield an antiserum to the cancer-associated phosphoprotein. Alternatively, a monoclonal antibody preparation containing antibodies to the cancer-associated phosphoprotein can be prepared by introducing into the bloodstream of a mouse the purified cancer-associated phosphoprotein preparation, allowing this preparation to remain in the mouse for at least about one day, removing spleen and/or lymph node cells from the mouse, fusing these removed cells with mouse myeloma cells and culturing the resultant hybridoma cells, selecting one or more hybridoma cells capable of producing the desired antibody and harvesting this antibody from the selected cell or cells. The antibody preparation produced by either of these techniques is substantially free of antibodies to normal plasma fraction, is capable of being precipitated by 35% saturated aqueous ammonium sulfate solution and is capable of forming a conjugate with the cancer-associated phosphoprotein. The antibody preparation is capable of forming a visible precipitate with the cancer-associated phosphoprotein when they are diffused towards one another in agar gel but is not capable of forming a conjugate with the 25K protein from human plasma nor the 35K protein fraction from rat plasma.
Finally, the aforementioned application Ser. No. 554,439 discloses that the presence of any of a wide variety of cancers can be detected in mammals by producing an antibody to the form of the cancer-associated phosphoprotein present in cancerous mammals of the appropriate species by the techniques discussed above, contacting these antibodies with biological material, preferably plasma, from the mammal, and detecting the presence of a reaction product between the antibodies and the antigen.
Obviously, this technique can be used to test a mammal for the presence of tumors which have been induced as a result of the exposure of the mammal to a carcinogen. However, it has now been discovered that not only can the test for the presence of the cancer-associated phosphoprotein detect the presence of tumors induced by carcinogens; in addition, this test will give positive results in mammals exposed to a carcinogen before macroscopically visible tumors are present in the mammal. Accordingly, this test provides a basis for a rapid carcinogen screening test.